by Cheng Sze Lok, Alfred. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 104-109). / Abstract also in Chinese. / DECLARATION --- p.i / ABSTRACT --- p.ii / ACKNOWLEDGEMENT --- p.vii / ABBREVIATIONS --- p.ix / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xii / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER ONE 226}0ؤ --- INTRODUCTION / Chapter 1.1 --- The Growth Plate / Chapter 1.1.1 --- "Function, Structure and Biochemistry of the Growth Plate" --- p.1 / Chapter 1.1.2 --- Extracellular Matrix of the Growth Plate Cartilage --- p.4 / Chapter 1.1.3 --- Vascular Supply to the Growth Plate --- p.9 / Chapter 1.1.4 --- Endochondral Ossification --- p.10 / Chapter 1.2 --- Growth Plate Damage and the Contemporary Reconstruction Models --- p.13 / Chapter 1.3 --- The 3-D Chondrocyte Pellet Culture --- p.15 / Chapter 1.4 --- The Study Plan --- p.16 / Chapter 1.5 --- The Objectives of the Study --- p.18 / Chapter CHAPTER TWO 一 --- METHODOLOGY / Chapter 2.1 --- Biosynthesis of Artificial Growth Plate using 3-D Chondrocyte Pellet Culture / Chapter 2.1.1 --- Isolation of Rabbit Costal Resting Chondrocytes --- p.19 / Chapter 2.1.2 --- Chondrocyte Monolayer Culture --- p.20 / Chapter 2.1.3 --- Three-dimensional Chondrocyte Pellet Culture --- p.20 / Chapter 2.1.4 --- Optimization of 3-D Chondrocyte Pellet Culture System --- p.20 / Chapter 2.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture / Chapter 2.2.1 --- Histomorphology --- p.22 / Chapter 2.2.2 --- Alkaline Phosphatase Histochemistry --- p.22 / Chapter 2.2.3 --- Collagen Typing --- p.23 / Chapter 2.2.3.1 --- Labeling and extraction of newly synthesized collagen / Chapter 2.2.3.2 --- SDS-PAGE and autoradiography / Chapter 2.2.4 --- Growth Rate --- p.25 / Chapter 2.2.4.1 --- Total DNA content determination / Chapter 2.2.4.2 --- Thymidine incorporation assay / Chapter 2.3 --- Implantation of Artificial Growth Plate and Assessment / Chapter 2.3.1 --- Implantation of Artificial Growth Plate into Partial Growth Plate Defect Model --- p.27 / Chapter 2.3.1.1 --- Animals / Chapter 2.3.1.2 --- Surgical procedure / Chapter 2.3.1.3 --- Experimental groups / Chapter 2.3.2 --- Histology --- p.30 / Chapter 2.3.3 --- Metabolism of Artificial Growth Plate In Vivo --- p.31 / Chapter 2.3.3.1 --- Radio sulfate labeling / Chapter 2.3.3.2 --- Liquid emulsion and autoradiography / Chapter CHAPTER THREE 一 --- RESULTS / Chapter 3.1 --- Biosynthesis of Artificial Growth Plate using 3-D Chondrocyte Pellet Culture / Chapter 3.1.1 --- Morphology of the Isolated Rabbit Chondrocyte --- p.32 / Chapter 3.1.2 --- Three-dimensional Chondrocyte Pellet Culture --- p.32 / Chapter 3.1.3 --- Optimization of 3-D Chondrocyte Pellet Culture System --- p.35 / Chapter 3.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture / Chapter 3.2.1 --- Histomorphology --- p.38 / Chapter 3.2.2 --- Alkaline Phosphatase Histochemistry --- p.43 / Chapter 3.2.3 --- Collagen Typing --- p.47 / Chapter 3.2.4 --- Growth Rate --- p.50 / Chapter 3.2.4.1 --- Total DNA content determination / Chapter 3.2.4.2 --- Thymidine incorporation assay / Chapter 3.3 --- Implantation of Artificial Growth Plate and Assessment / Chapter 3.3.1 --- Histology --- p.54 / Chapter 3.3.2 --- Metabolism of Artificial Growth Plate In Vivo --- p.65 / Chapter CHAPTER FOUR 一 --- DISCUSSION / Chapter 4.1 --- Optimal Condition for 3-D Chondrocyte Pellet Culture System --- p.67 / Chapter 4.1.1 --- Some Critical Characteristics of the Growth Plate --- p.68 / Chapter 4.1.2 --- Selection of Animal Model --- p.69 / Chapter 4.1.3 --- Optimization of Culturing Conditions 226}0ؤ Screening Based on Morphological Studies --- p.69 / Chapter 4.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture --- p.73 / Chapter 4.2.1 --- Development of the 3-D Chondrocyte Pellet Culture --- p.73 / Chapter 4.2.2 --- Development of the Chondrocyte Monolayer Culture --- p.78 / Chapter 4.2.3 --- Comparing the 3-D Chondrocyte Pellet Culture and Monolayer Culture --- p.79 / Chapter 4.2.3.1 --- Cellular organization / Chapter 4.2.3.2 --- Terminal differentiation of chondrocytes / Chapter 4.2.3.3 --- Cell division potential / Chapter 4.2.3.4 --- Production of cartilaginous matrix / Chapter 4.3 --- Resumption of Physeal Characteristics by Artificial Growth Plate In Vivo --- p.86 / Chapter 4.3.1 --- Three Stages of In Vivo Development of the Artificial Growth Plate --- p.86 / Chapter 4.3.1.1 --- Incorporation of artificial growth plate with host tissues / Chapter 4.3.1.2 --- Growth of the artificial growth plate invivo / Chapter 4.3.1.3 --- Resumption of endochondral ossification in the artificial growth plate / Chapter 4.3.2 --- Significance of Development of the 3-D Pellet Culture on its In Vivo Development --- p.89 / Chapter 4.3.2.1 --- 3-D pellet culture processes similar extracellular matrix with host / Chapter 4.3.2.2 --- 3-D pellet culture acquires growth plate-like cellular organization and differentiation pattern / Chapter 4.3.3 --- Effect of Host Microenvironment on Artificial Growth Plate Development --- p.90 / Chapter 4.3.3.1 --- Orientation of artificial growth plate implants / Chapter 4.3.3.2 --- Evidence from development of 3-D pellet culture in longer period of culture / Chapter 4.4 --- Comparison with other Growth Plate Reconstruction Models --- p.93 / Chapter 4.4.1 --- Implantation of Biologic or Inert Fillers --- p.93 / Chapter 4.4.2 --- Physeal Transplantation --- p.94 / Chapter 4.4.3 --- Transplantation of Cartilage Allografts --- p.95 / Chapter 4.4.4 --- Transplantation of High-density Chondrocyte Culture --- p.96 / Chapter CHAPTER FIVE 一 --- SUMMARY AND CONCLUSION --- p.98 / Chapter CHAPTER SIX 一 --- FURTHER STUDIES --- p.102 / REFERENCES --- p.104

Articular cartilage is a mechanically important soft tissue whose organisation at the micro- and nanoscale is critical for healthy joint function and where degeneration is associated with widespread disorders such as osteoarthritis. The tissue possesses a complex, graded and depth-dependent structure and at the nanoscale, cartilage mechanical functionality is dependent on the collagen and hydrated proteoglycans that form the extracellular matrix. The structure and in situ dynamic response of the collagen fibrils at the nanoscale, however, remain unclear. Here we utilise small angle X-ray diffraction to measure the depth-wise structure of the fibrillar architecture whilst performing time-resolved measurements during compression of bovine and human cartilage explants. We demonstrate the existence of a depth-dependent fibrillar pre-strain as determined by the D-periodicity, estimated at approximately 1-2%, due to osmotic swelling pressure from the proteoglycans. Furthermore, we reveal a rapid reduction and recovery of this pre-strain during stress relaxation, approximately 60 seconds after onset of peak load. Selective proteoglycan removal disrupts both collagen fibril pre-strain and transient responses during stress relaxation. Additionally, we show that IL-1β induced tissue inflammation also results in a reduction in fibrillar pre-strain and altered fibrillar mechanics. Cyclic loading induces a dynamic reduction and recovery in the D-period that is present regardless of loading rate or treatment, along with changes in diffraction peak intensities and widths. These findings suggest that the fibrils respond to loading via intra- and inter-fibrillar disordering alongside a transient response that is mediated by changes in hydration. These are the first studies to highlight previously unknown transient and cyclic responses to loading at the fibrillar level, and are likely to transform our understanding of the role of collagen fibril nano-mechanics in cartilage and other hydrated soft tissues. These methods can now be used to better understand cartilage in aging and other muscoskeletal diseases.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease which affects the joint structures leading to disability. Studies in the last 20 years have documented the increased prevalence of knee pain and symptomatic knee OA. Similarly, of temporomandibular joint (TMJ) disorders OA is the most common. Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. previous studies showed that LOXL2 is elevated during mouse fracture healing. Our hypothesis that LOXL2 acts as a specific anabolic factor in chondrocytes METHODS: The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. We also examined if LOXL2 induces the proliferation of OA chondrocytes. RESULTS: LOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-β1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1β and TNF-α downregulated LOXL2, in human chondrocytes. LOXL2 expression also inhibited IL-1β-induced phospho-NF-κB/p65 and TGF-β1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. We have found that LOXL2 does not induce the proliferation of human TMJ or knee OA chondrocytes. CONCLUSIONS: We showed that LOXL2 induces differentiation and attenuates OA related catabolic signaling pathways.

Cheung Wing-hoi. / "December 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Bone formation occurs through two ossification processes, intramembranous and endochondral. Intramembranous ossification is characterized by the direct differentiation of stem cells into osteoblasts, which then create bone. Endochondral ossification involves an intermediate step, as stem cells first differentiate into chondrocytes and produce a cartilage anlage. The chondrocytes mature into hypertrophic chondrocytes, which transform the cartilage anlage into bone. Bone tissue engineering has predominantly mimicked intramembranous ossification, creating osteoblast-based grafts through the direct differentiation of stem cells. Though successful in specific applications, greater adoption of osteoblast-based grafts has failed due to incomplete integration, limited regeneration, and poor mechanical maintenance. To overcome these obstacles, inspiration was drawn from native bone fracture repair, creating tissue engineered bone grafts replicating endochondral ossification. Hypertrophic chondrocytes, the key cell in endochondral ossification, were differentiated from mesenchymal stem cell sources by first generating chondrocytes and then instigating maturation to hypertrophic chondrocytes. Conditions influencing this differentiation were investigated, indicating the necessity of prolonged chondrogenic cultivation and elevated oxygen concentrations to ensure widespread hypertrophic maturation. Comparing the bone production performance of differentiated hypertrophic chondrocytes to differentiated osteoblasts revealed that hypertrophic chondrocytes deposit significantly greater volume of bone mineral at a higher density than osteoblasts, albeit in a more juvenile form. When implanted subcutaneously, the hypertrophic chondrocytes stimulated turnover of this juvenile template into compact-like bone, whereas osteoblasts proceeded with processes similar to bone remodeling, generating spongy-like bone. Implanting these tissue engineered constructs into an orthotopic, critical-sized femoral defect saw hypertrophic chondrocyte-based constructs integrate quickly with the femur and facilitate the creation of significantly more bone, resulting in a successful bridging of the defect. The success of hypertrophic chondrocyte-based grafts in overcoming the failures of tissue engineered bone grafts demonstrates the potential of endochondral ossification inspired bone strategies and prompts its further investigation towards clinical utilization.

The articular cartilage has a limited capacity to regenerate. Cartilage lesions often result in degeneration, leading to osteoarthritis. Current treatments are mostly palliative and reparative, and fail to restore cartilage function in the long term due to the replacement of hyaline cartilage with fibrocartilage. Although a stem-cell based approach towards regenerating the articular cartilage is attractive, cartilage generated from human mesenchymal stem cells (hMSCs) often lack the function, organization and stability of the native cartilage. Thus, there is a need to develop effective methods to engineer physiologic cartilage tissues from hMSCs in vitro and assess their outcomes in vivo. This dissertation focused on three coordinated aims: establish a simple in vivo model for studying the maturation of osteochondral tissues by showing that subcutaneous implantation in a mouse recapitulates native endochondral ossification (Aim 1), (ii) develop a robust method for engineering physiologic cartilage discs from self-assembling hMSCs (Aim 2), and (iii) improve the organization and stability of cartilage discs by implementing spatiotemporal control during induction in vitro (Aim 3). First, the usefulness of subcutaneous implantation in mice for studying the development and maintenance of osteochondral tissues in vivo was determined. By studying juvenile bovine osteochondral tissues, similarities in the profiles of endochondral ossification between the native and ectopic processes were observed. Next, the effects of extracellular matrix (ECM) coating and culture regimen on cartilage formation from self-assembling hMSCs were investigated. Membrane ECM coating and seeding density were important determinants of cartilage disc formation. Cartilage discs were functional and stratified, resembling the native articular cartilage. Comparing cartilage discs and pellets, compositional and organizational differences were identified in vitro and in vivo. Prolonged chondrogenic induction in vitro did not prevent, but expedited endochondral ossification of the discs in vivo. Finally, spatiotemporal regulation during induction of self-assembling hMSCs promoted the formation of functional, organized and stable hyaline cartilage discs. Selective induction regimens in dual compartment culture enabled the maintenance of hyaline cartilage and potentiated deep zone mineralization. Cartilage grown under spatiotemporal regulation retained zonal organization without loss of cartilage markers expression in vivo. Instead, cartilage discs grown under isotropic induction underwent extensive endochondral ossification. Together, the methods established in this dissertation for investigating cartilage maturation in vivo and directing hMSCs towards generating physiologic cartilage in vitro form a basis for guiding the development of new treatment modalities for osteochondral defects.

There are limited treatment options available today to slow down progression of osteoarthritis in its early stages and most interventions, such as highly invasive partial and total joint replacement surgeries, are performed only at the late stages of the disease. Understanding the mechanism of early articular cartilage stress-mediated wear and failure can aid in the design of new treatment options that are introduced at earlier stages of the disease, presenting the potential to slow down osteoarthritis progression and thus significantly improve patient outcomes. This dissertation aims to provide a basic science understanding of wear propagation and repair of articular cartilage in the absence of traumatic events under the normal reciprocal sliding motion of the articular layers at physiologic load magnitudes. In this dissertation there are three main thrusts: (1) characterize cartilage delamination wear under normal sliding (2) define a chemical environment that promotes cartilage explant homeostasis to enable long-term wear-and-repair studies (3) investigate a practical treatment modality capable of stopping or slowing down structural degeneration of articular cartilage in OA. We hypothesize that the mode of cartilage damage is delamination wear that progresses by fatigue failure of the extracellular matrix (ECM) under physiologic sliding, even when cartilage layers are subject to physiologic load magnitudes and contact stresses and even when the friction coefficient μ remains low (H1a). Based on prior literature findings regarding the role of synovial fluid (SF) boundary lubricants on the reduction of friction and wear, we also test the hypothesis (H1b) that SF delays the onset of cartilage delamination when compared to physiological buffered saline (PBS). We then test a third hypothesis (H1c) that loading cartilage against cartilage delays the onset of delamination wear compared to testing glass on cartilage, since contacting porous cartilage layers exhibit a much smaller solid-on-solid contact area fraction than impermeable glass contacting porous cartilage. Next, we hypothesize that the homeostatic dysregulation previously observed in cultured immature cartilage explants results from the presence of non-physiologic levels of important metabolic mediators in the culture medium. To this end, we hypothesize that: (H2a) immature bovine cartilage explants cultured in native synovial fluid will maintain homeostasis as characterized by maintenance of their mechanical properties and ECM contents at initial (post- explantation) levels, and (H2b) explants cultured in a physiologic-based medium, consisting of physiologic levels of key metabolic mediators, will maintain a similar homeostasis over long- term culture. Finally, a laser treatment strategy is explored that has the capability to reform collagen crosslinks, replacing those lost during OA progression. This novel therapy acts without injuring the cells and without any chemical additive or thermal ablation. The laser treatment protocol used in this application can specifically target the subsurface region, located 200 μm of the articular surface. By strengthening this region with enhanced crosslinking, we hypothesize (H3a) that cartilage will demonstrate greater resistance to fatigue failure than untreated controls. We then hypothesized (H3b) that this treatment protocol would also be effective on devitalized fibrillated human articular cartilage from OA joints with overall Outerbridge score OS1-3. We find that for both cartilage-on-cartilage and glass-on-cartilage sliding configurations at physiologic applied loads, long-term sliding with a low friction coefficient causes wear in the form of delamination. We show that the use of synovial fluid as a lubricant delays the onset of wear; and, similarly, that sliding with a cartilage counterface also reduces the incidence of wear. In subsequent studies we fully characterize a homeostatic culture medium to emulate cartilage in vitro behavior in synovial fluid. We show that explants cultured in this medium can maintain their properties for at least one month and have no loss in cell viability. Laser treatment is then tested on both living and devitalized bovine and devitalized human cartilage and the treatment is shown to improve the wear resistance of the tissue without harming embedded cells. Overall this work has led to novel insights that have clinical applicability. One strength of the in vitro investigations described in this body of work is the ability to separate out mechanically-mediated events from biochemically-mediated events, which would be impossible in vivo. Parsing out such specific mechanisms of cartilage wear can help guide better understanding of disease progression and drive therapeutic intervention. Intervening during the early stages of OA offers the promise of preventive care that currently does not exist and could provide significant benefits to a patient’s quality of life. This dissertation asserts that focusing on delaying or preventing wear by improving the resiliency of the extant intact cartilage in early OA is a viable strategy to improve patient outcomes and offers an innovative approach over existing regenerative techniques.

Damaged cartilage can cause severe pain and restricted mobility, with few long term treatments available. The developing field of tissue engineering offers an alternative to the currently used full joint replacement. Restoring damaged cartilage through tissue engineering would enable an active lifestyle to be recovered and retained, without restrictions to joint mobility. This is increasingly important as the prevalence of osteoarthritis rises. Tissue engineering requires biomaterial scaffolds that mimic the function of the tissue while cells develop, and so the scaffold must provide the appropriate biological, chemical and mechanical stimuli. In this work, methods were developed to enable the design of scaffolds that mimic the microstructure and mechanical properties of articular cartilage. Electrospinning was investigated as a method to mimic the nanoscale collagen fibres within cartilage extracellular matrix. A parametric study was conducted to determine how changes to a gelatin solution affect the mechanical properties of the non-woven fibrous mesh. The solution properties had a clear impact on the morphology of the fibres, but the effect on the mesh mechanical properties was convoluted. The results demonstrated the need for greater understanding of the 3D morphology of electrospun meshes, to establish how these may be altered in order to design scaffolds with desirable mechanical properties. The fracture mechanics of soft materials are complex, and are generally overlooked when designing tissue engineering scaffolds. The complexities have led to a lack of standardised testing, making comparisons between studies impractical. In this work, fracture testing methods were compared, using a viscoelastic polymer to mimic some of the complexities of soft tissue mechanics. Mode III trouser tear tests and mode I pure shear tests were found to provide reliable measurements. Due to the ease of testing small samples, trouser tear testing was concluded to be the most advantageous for determining the fracture resistance of soft tissue engineering scaffolds. Finally, electrospun meshes were combined with hydrogels to create biomimetic scaffolds, which were characterised using tensile and trouser tear fracture tests. Fibre-reinforcement was shown to enhance the mechanical properties of a weak hydrogel, but diminished those of a strong, tough polyacrylamide (PAAm)-alginate hydrogel. The PAAm-alginate hydrogel exhibited mechanical properties close to those of natural articular cartilage, but without the microstructure that would enhance its suitability for use as a cartilage tissue engineering scaffold. An alternative method for reinforcing PAAm-alginate was proposed, which shows promise for producing a biocompatible scaffold that mimics both the mechanics and the microstructure of articular cartilage. Ultimately, this thesis aimed to improve the design of biomimetic scaffolds for cartilage tissue engineering, and advance mechanical characterisation techniques within this field.

Articular cartilage is the connective tissue lining the ends of long bones, providing a dynamic surface that bears load while providing a smooth surface for articulation. When damaged, however, this tissue exhibits a poor capacity for repair, lacking the lymphatics and vasculature necessary for remodeling. Osteoarthritis (OA), a growing health and economic burden, is the most common disease afflicting the knee joint. Impacting nearly thirty million Americans and responsible for approximately $90 billion in total annual costs, this disease is characterized by a progressive loss of cartilage accompanied by joint pain and dysfunction. Moreover, while generally considered to be a disease of the elderly (65 years and up), evidence suggests the disease may be traced to joint injuries in young, active individuals, of whom nearly 50% will develop signs of OA within 20 years of the injury. For these reasons, significant research efforts are directed at developing tissue-engineered cartilage as a cell-based approach to articular cartilage repair. Clinical success, however, will depend on the ability of tissue-engineered cartilage to survive and thrive in a milieu of harsh mechanical and chemical agents. To this end, previous work in our laboratory has focused on growing tissues appropriate for repair of focal defects and entire articular surfaces, thereby investigating the role of mechanical and chemical stimuli in tissue development. While we have had success at producing replacement tissues with certain qualities appropriate for clinical function, engineered cartilage capable of withstanding the full range of insults in vivo has yet to be developed. For this reason, and in an effort to address this shortcoming, the work described in this dissertation aims to (1) further characterize and (2) optimize the response of tissue-engineered cartilage to physical loading and the concomitant chemical insult found in the injured or diseased diarthrodial joint, as well as (3) provide a clinically relevant strategy for joint resurfacing. Together, this holistic approach maximizes the chances for in vivo success of tissue-engineered cartilage. Regular joint movement and dynamic loads are important for the maintenance of healthy articular cartilage. Extensive work has been done demonstrating the impact of mechanical load on the composition of the extracellular matrix and the biosynthetic activity of resident chondrocytes in explant cultures as well as in tissue-engineered cartilage. In further characterizing the response of tissue-engineered cartilage to mechanical load, the work in this dissertation demonstrated the impact of displacement-controlled and load-controlled stimulation on the mechanical and biochemical properties of engineered cartilage. Additionally, these studies captured tension-compression nonlinearity in tissue-engineered cartilage, highlighting the role of the proteoglycan-collagen network in the ability to withstand dynamic loads in vivo, and optimized a commercial bioreactor for use with engineered cartilage. The deleterious chemical environment of the diseased joint is also well investigated. It is therefore essential to consider the impact of pro-inflammatory cytokines on the mechanical and biochemical development of tissue-engineered cartilage, as chemical injury is known to promote degradation of extracellular matrix constituents and ultimately failure of the tissue. Combining expertise in interleukin-1\alpha, dexamethasone, and drug delivery systems, a dexamethasone drug delivery system was developed and demonstrated to provide chondroprotection for tissue-engineered cartilage in the presence of supraphysiologic doses of pro-inflammatory cytokines. These results highlight the clinical relevance of this approach and indicate potential success as a therapeutic strategy. Clinical success, however, will not only be determined by the mechanical and biochemical properties of tissue-engineered cartilage. For engineered cartilage to bear loads in vivo, it is necessary to match the natural topology of the articular surface, recapitulating normal contact geometries and load distribution across the joint. To ensure success, a method for fabricating a bilayered engineered construct with biofidelic cartilage and subchondral bone curvatures was developed. This approach aims to create a cell-based cartilage replacement that restores joint congruencies, normalizes load distributions across the joint, and serves as a potential platform for the repair of both focal defects and full joint surfaces. The research described in this dissertation more fully characterizes the benefits of mechanical stimulation, prescribes a method for chondroprotection in vivo, and provides a strategy for creating a cartilage replacement that perfectly matches the native architecture of the knee, thus laying the groundwork for clinical success of tissue-engineered cartilage.

Osteoarthritis (OA) is a debilitating degenerative joint disease affecting 27 million Americans over the age of 25. Whereas OA is a disease of the entire joint organ, the contribution of the synovium, a specialized lining that envelops the knee joint, to cartilage degeneration and disease progression has been underappreciated. Synovial inflammation often precedes the development of cartilage damage and is observed in early and late stage OA. The onset of synovitis is driven by both elevated concentrations of pro-inflammatory cytokines and tissue debris in the joint space. Accordingly, surgeons have observed cartilaginous debris embedded within the synovium of OA patients presenting with severe synovial hyperplasia. It has been hypothesized that the fibrotic shortening of the synovial capsule results in OA pain and joint stiffness and contributes to further joint destruction through the release of degradative enzymes. Current strategies to treat synovial inflammation and joint pain, such as intra-articular injections and synovectomy, have had limited and variable success. To this end, cell and tissue engineering culture models provide a versatile platform to study the tissues and cells involved in OA. Our lab has typically employed mechanical overload or cytokine insult of chondrocytes and cartilage explants to study cartilage degradation. Similarly, to isolate the role of synovium in OA, synovial explants or fibroblast-like synoviocytes (FLS) can be exposed to chemical or physical OA stimuli. Although often overlooked as an instigator of OA, cartilage wear particles have been reported to induce synovial inflammation and OA-like joint changes in various animal models. As opposed to non-biologic (metal or plastic) wear particles, small (sub-10um) cartilage wear particles are comprised of extracellular matrix constituents that are degradable and may interact with cells beyond phagocytosis. Using cells derived from the pathologic joint provides the opportunity to study inherent changes to OA cells (both FLS and chondrocytes) within their own de novo extracellular matrix. The work presented in this dissertation aims to combine knowledge from basic science and pre-clinical culture models of OA to develop a clinically relevant disease model using cells derived from clinical samples.